Whole Genome CRISPR Screening Strategy to Identify Genes Contributing to
SARS-CoV-2 Spike and VSV-G Mediated Entry.
Abstract
Background Understanding the cellular host factors that promote
and inhibit viral entry is important for identifying viral
countermeasures. CRISPR whole genome screens can be used to rapidly
discover host factors that contribute to or impair viral entry. However,
when using the live viruses and cellular lethality for selection, these
screens can identify an overwhelming number of genes without specificity
for the stage of the viral infection cycle. New screening methods are
needed to identify host machinery contributing to specific steps of
viral infection. Here, we developed a CRISPR whole genome screen and
counter screen strategy based on a pseudoviral platform that allowed
identification of genes specific to SARS-CoV-2 spike and vesicular
stomatitis virus glycoprotein VSV-G mediated entry. Methods To
focus the screen onto the entry step, we used non-lytic fluorescent
reporters in combination with a comparative counter screen strategy to
distinguish host genes affecting the pseudoviral reporter from those
unique to envelope-mediated entry. Screening of SARS-CoV-2 spike and
VSV-G on the same lentiviral pseudovirus allowed identification of
entry-specific genes relative to genes associated with
retro-transcription, integration, and reporter expression from the
lentiviral pseudovirus. Second, a Cre-Gag fusion protein in the
pseudovirus was used to bypass retro-transcription and integration by
directly activating a floxed GFP reporter upon entry to reduce the
number of gene hits and increase specificity for viral entry.
Results Our approach correctly identified SARS-CoV-2 and VSV-G
receptors ACE2 and LDLR, respectively and distinguished genes associated
with retroviral reporter expression from envelope-mediated entry.
Moreover, the CRE-Gag fusion/flox reporter increased the screen
specificity for viral entry associated genes. Validation of a few hits
demonstrates that this approach distinguishes envelope-specific host
factors from genes affecting reporter expression. Conclusion
Overall, this approach provides a new strategy for identifying host
genes influencing viral entry without the confounding complexity of
live-viral screens which produce long gene lists associated with all
aspects of viral pathogenesis and replication. This approach provides a
pathway for increasing the specificity of CRISPR whole genome screens
for identifying host genes contributing to specific steps in viral
infection.